Re: [ccp4bb] Protein concentration vs Molecular wt...

[Patrick Shaw Stewart]

See  http://www.douglas.co.uk/PDB_data.htm, section 4


On 19 July 2012 20:58, james09 pruza  wrote:

> Dear Crystallographers,
> Is there any rule of thumb for Protein concentration and molecular weight
> for crystallization trials of a soluble protein? Looking for high molecular
> wt. protein ~50kDa.
> James.
>
>


-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
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[ccp4bb] Job advert: Research Fellow at the University of Leicester, Structural Studies of Protein-Drug Interactions

[Bayliss, Richard (Dr.)]

A position is available in the Centre for Translational Therapeutics (CTT) 
within the College of Medicine, Biological Sciences and Psychology for a 
talented postdoctoral structural biologist to work on a collaborative in-house 
structural biology projects, with an emphasis on structure-based drug discovery 
and development. The CTT is headed by Prof. Andrew Tobin and aims to facilitate 
early phase drug discovery within the College. The scientist will be based in 
the Department of Biochemistry and will work alongside the groups of Prof. Mark 
Carr (NMR spectroscopy) and Dr Richard Bayliss (X-ray crystallography), who 
have strong track records in structure-based drug discovery. The scientist will 
determine the structures of proteins that are targeted by CTT and characterise 
their interactions with candidate compounds. The proposed work will involve 
close interactions with other postdoctoral scientists within the CTT, research 
groups across the College and medicinal chemistry collaborators. The CTT and 
Department of Biochemistry are superbly equipped for inhibitor discovery using 
mobility-shift, label-free and thermal shift assays, as well as for all aspects 
of NMR spectroscopy and X-ray crystallography based structural biology, with 
excellent supporting facilities for recombinant protein expression and 
purification.

The successful postdoctoral research fellow will have a proven track record in 
structural biology, with significant experience in X-ray crystallography and/or 
NMR-based approaches. A willingness to apply either approach to solve protein 
structures would be desirable. A strong interest in knowledge-based drug 
discovery is essential. The ability and willingness to travel to meetings with 
collaborators and to use off-campus research facilities is essential. Good 
communication and presentation skills are also a pre-requisite.

Salary Grade 8 - £39,257 to £44,166 per annum. Available immediately for an 
initial period of 1 year, with the expectation of extension for a further 2 
years

Informal enquiries may be made to Dr Richard Bayliss, Tel +44 (0)116 229 7100, 
E.mail richard.bayl...@le.ac.uk; or Prof. Mark Carr, Tel: +44 (0)116 229 7075, 
E.mail: md...@le.ac.uk; or Prof. Andrew Tobin, Tel: +44 (0)116 252 2935, Email: 
t...@le.ac.uk

Applications must be made through the University jobs page, on which further 
details are available 
(http://www2.le.ac.uk/offices/jobs/opportunities/jobsearch - reference MBP00666)

The closing date for this post is midnight on 19th August 2012. 

=
Dr Richard Bayliss, Reader in Structural Biology
Department of Biochemistry
Henry Wellcome Building
University of Leicester
Lancaster Road, Leicester
LE1 9HN

Tel: 0116 2297100
Web: 
http://www2.le.ac.uk/departments/biochemistry/staff/richard-bayliss/research

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[ccp4bb] advice on spectrometric instruments choice

[Sebastiano Pasqualato]

Dear all,
I'm looking for some advices on the choice of some spectroscopic equipment for 
our lab.

What we would like to have is the possibility of measuring Circular Dichroism 
spectra, and perform kinetic analysis in fluorescence, possibly also with a 
stopped-flow set up, for pre-steady state kinetics.

Would you reckon a single instrument would be capable of fulfilling these 
tasks, or would you rather advice the investment on two instruments?

Could you guys please give me suggestions on piece of instruments that you're 
happy (or unhappy) with?
I've been currently considering the Chirascan from Applied Photophysics, or the 
Jasco J-815, so far. Any comment on those?

Thanks a lot in advance,
best regards,
ciao,
s

-- 
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990

please note the change in email address!
sebastiano.pasqual...@ieo.eu

Re: [ccp4bb] advice on spectrometric instruments choice

[Scott Thomas Walsh]

Hi Sebastiano,

We have a Chirascan instrument with all the attachments (fluorescence, stopped 
flow, multi-cell attachment, etc).  It is by far the best CD 
instrument that I have used.  I have experience in the past with Aviv, Jasco, 
and Olis instruments.

Cheers,

Scott


Scott T. R. Walsh, Ph.D.
Assistant Professor
University of Maryland
IBBR/CBMG
Rm 3127E CARB-2
9600 Gudelsky Drive
Rockville, MD 20850
email: swals...@umd.edu
phone: (240) 314-6478
fax: (240) 314-6255

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sebastiano 
Pasqualato [sebastiano.pasqual...@gmail.com]
Sent: Friday, July 20, 2012 8:01 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] advice on spectrometric instruments choice

Dear all,
I'm looking for some advices on the choice of some spectroscopic equipment for 
our lab.

What we would like to have is the possibility of measuring Circular Dichroism 
spectra, and perform kinetic analysis in fluorescence, possibly also with a 
stopped-flow set up, for pre-steady state kinetics.

Would you reckon a single instrument would be capable of fulfilling these 
tasks, or would you rather advice the investment on two instruments?

Could you guys please give me suggestions on piece of instruments that you're 
happy (or unhappy) with?
I've been currently considering the Chirascan from Applied Photophysics, or the 
Jasco J-815, so far. Any comment on those?

Thanks a lot in advance,
best regards,
ciao,
s

--
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990

please note the change in email address!
sebastiano.pasqual...@ieo.eu

Re: [ccp4bb] Nucleophilic attack by the side-chain carboxyl group of Asp?

[Roger Rowlett]

Asp is not a strong nucleophile, and coordination to zinc will make it less
so. It is more likely that a zinc-bound water is acting as a nucleophile.
If there is no observed water attached to your Zn, consider the possibility
of ligand-water exchange, as in bacterial beta-carbonic anhydrases.

Roger Rowlett
On Jul 20, 2012 1:31 AM, "Crystal Xu"  wrote:

> Hi everyone,
>
> I am studying the catalytic mechanism of an enzyme. My structural data
> indicates that an Asp is of most possibility to sever as a general base.
> The Asp residue is coordinated to a zinc ion. Would it be possible that the 
> side-chain
> carboxyl group of Asp attacks a carbon atom of the substrate? Does anyone
> know any examples?
>
> Thanks very much.
>
> Best regards.
>
>
>

[ccp4bb] EDS server woes resolved

[Gerard DVD Kleywegt]

Dear all,

The EDS server was unfortunately down for a number of days. However, as of 
yesterday it is up and running again. Apologies for the inconvenience.


  http://eds.bmc.uu.se/

--Gerard

**
   Gerard J. Kleywegt

  http://xray.bmc.uu.se/gerard   mailto:ger...@xray.bmc.uu.se
**
   The opinions in this message are fictional.  Any similarity
   to actual opinions, living or dead, is purely coincidental.
**
   Little known gastromathematical curiosity: let "z" be the
   radius and "a" the thickness of a pizza. Then the volume
of that pizza is equal to pi*z*z*a !
**

Re: [ccp4bb] Protein concentration vs Molecular wt...

[Oganesyan, Vaheh]

Number of years ago Jaru Jancarik (the author of Screen I & II sold by HR) 
while in Berkeley Structural Genomics Center (or may be even earlier) made an 
observation regarding protein precipitation in condition A6 in that very 
screen. Based on this observation HR sells now PCT (protein concentration test 
or something similar). In brief, if your protein is concentrated enough and is 
a regular protein, not a stellar, you will see medium to heavy precipitation. 
In the case of proteins that are really of great quality you'll end up having 
crystals in A6. After many years of experience I realized that A9 and A10 from 
the same screen could be added to the list of conditions that are somewhat 
indicative for choosing right concentration same way like Jaru did for A6.

My two Armenian drams worth,

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger 
Rowlett
Sent: Thursday, July 19, 2012 4:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein concentration vs Molecular wt...

This is almost exactly our basic approach, too. Before we got a dropsetter, we 
did 24 wells (1/2 screen) to get a feel for the correct protein concentration. 
Some additional rules of thumb we use:

  1.  We usually start at 10 mg/mL protein and go up or down from there 
depending on the results of the initial screen
  2.  If we observe a high frequency of precipitation in a 10 mg/mL protein 
screen, we will usually set a 1/2 concentration screen by diluting the screen 
solutions 1:1 with water. This frequently uncovers additional hits in wells 
that were heavily precipitated in the original screen. Empirically, proteins we 
study seem to crystallize better in the higher protein/lower precipitant zone 
of the phase diagram than the lower protein/higher precipitant zone. YMMV.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 7/19/2012 4:31 PM, mjvdwo...@netscape.net 
wrote:
I don't think there is such a rule, but in the old days, when we only had 
Hampton Screen I and II, the rule was:

- Set up screen 1, look at the drops and you should expect some kind of 
precipitation in 50% of the drops. If much less than that, increase your 
protein concentration. If much more than that, decrease protein concentration.
- Set up screen 2, look and expect 30% precipitation.

I used to cut corners and do the statistics at 1/2 of a screen (one 24-well 
plate). You can probably use this method to get within a factor of 2 of the 
optimal concentration.

There are probably good statistics in the papers for the screens that you may 
use. One of the advantages of structural genomics efforts is that these things 
are known (and hopefully published).

Even older trick is to take a drop of protein and look under a microscope, 
record how much AmSO4 it takes to cause precipitation. Do the same with PEG. 
Keep adding a little at a time and look immediately. This will give you an idea 
if you are near a reasonable concentration. I think that this latter method 
does not tell you much more than "physics-information" - which is how many 
zeroes there are: whether 1 mg/ml or 10mg/ml or 100mg/ml is reasonable.

Mark

-Original Message-
From: james09 pruza 
To: CCP4BB 
Sent: Thu, Jul 19, 2012 1:59 pm
Subject: [ccp4bb] Protein concentration vs Molecular wt...
Dear Crystallographers,
Is there any rule of thumb for Protein concentration and molecular weight for 
crystallization trials of a soluble protein? Looking for high molecular wt. 
protein ~50kDa.
James.

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Re: [ccp4bb] off topic Thermal shift assay

[Dr. Lorenzo Finci]

Noor, 
Thank you very much for your inquiry. As we all know, thermodynamic principles 
of cooperativity and allostery have long been used as a foundation to begin 
understanding the complex interplay between associated ligand binding events. 
In principle, the delta Tm shifts that occur when multiple ligands bind to the 
same protein should further manifest cooperative effects between the inherent 
binding sites. A unique property attributed to the Thermofluor is that it 
offers a high throughput approach to the study of allosteric interactions 
between protein and ligand. In terms of unfolding, Thermofluor has the 
capability to answer whether the flexibility of the protein is expressed as the 
number of different stable conformational states in high or low quantities. 
This is due to the fact that the range of temperature that unfolding occurs is 
reported by the flexibility of the protein. For example, steep transitions are 
indicative of highly cooperative unfolding, whereas shallow transitions 
indicate high flexibility. Multidomain proteins reflect an observed monophasic 
unfolding transition, and this is what is generally accepted as two-state 
unfolding. More complex unfolding transitions reflect that unfolding of the 
domains does not occur in a concerted manner. In order to obtain a detailed 
understanding of the linkage between ligand binding and protein stability, a 
concert of biophysical characterization utilizing Thermofluor, ITC, and DSC 
should be utilized... 
Refferences:1. Binding Techniques to Study the Allosteric Energy Cycle; 
Allostery: Methods and Protocols, Methods in Molecular Biology, 2012, Kranz et 
al2. http://.thermofluor.org3. 
http://thermofluor.org/resources/Niesen-fingerprinting_Oxford.pdf4. 
Thermodynamic Stability of Carbonic ANhydrase: Measurements of Binding Affinity 
and Stoichiometry Using Thermofluor, Biochemistry, 2005, Matulis et al
Sincerely, lorenzo

Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard 
Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe 
College of Life SciencesBeijing, China


Date: Thu, 19 Jul 2012 21:23:59 +0100
From: mohamed.n...@ul.ie
To: lfi...@hotmail.com
Subject: Re: [ccp4bb] off topic Thermal shift assay


  

  
  
Dear Lorenzo



> a measure of protein cooperatively,



  Regarding
your comment on positive cooperativity, is there any literature
on this? I was taught that positive cooperativity for enzyme
will require a steady-state kinetic assay. How does this relate
to protein unfolding as measured by thermal shift?



Sorry for a basic question.



Thanks.



Regards

Mohamed



  
  Mohamed Noor

Chemical and Environmental Sciences Department

University of Limerick

Ireland



  
  On 19/07/2012 16:25, Dr. Lorenzo Finci wrote:



  

[ccp4bb] Ferredoxin Containing Crystal Bleaching

[RHYS GRINTER]

Hi All,

I was collecting some data at home on a crystal from a protein containing a 
ferredoxin domain. This crystal was originally red-brown in colour, but after 
16h of data collection, a large portion of it appears not be colourless. The 
crystal still seems to diffract fine (beyond 2A).
I assume this it due in some way to the radiation exposure but was wondering 
out curiosity whether someone had experienced this before and had a specific 
explanation for it. And if this will effect the integrity of the data.

Cheers

Rhys Grinter
PhD Candidate
University of Glasgow

Re: [ccp4bb] Ferredoxin Containing Crystal Bleaching

[Yuri Pompeu]

Actually with haeme, and or flavin containing enzymes it is known for the 
cofactor to undergo reduction.
There is a recent JACS paper that explores these changes as a function of X-ray 
exposure.
(2012) J.Am.Chem.Soc. 134: 2823-2834
Excellent work done in part at Allen Orville's X6A
As far as quality of your data goes, unless your crystal is suffering from 
radiation damage (they all do!) from those free electrons/radicals
then your data should not be affected by the bleaching.
HTH.
Yuri

Re: [ccp4bb] Ferredoxin Containing Crystal Bleaching

[Edward A. Berry]

Iron sulfur proteins tend to absorb less (although not zero) in the visible
when reduced. We see this with succinate dehydrogenase when we reduce with
dithionite to see the heme spectrum- the difference extinction coefficient
at the beta "peak" is actually negative because the baseline absorbance
due to three different Fe-S centers is bleached.
 (Acta Cryst. D61, 
380 Fig 2)    

Published spectra for soluble Rieske protein fragments also show bleaching.

Yuri Pompeu wrote:

Actually with haeme, and or flavin containing enzymes it is known for the 
cofactor to undergo reduction.
There is a recent JACS paper that explores these changes as a function of X-ray 
exposure.
(2012) J.Am.Chem.Soc. 134: 2823-2834
Excellent work done in part at Allen Orville's X6A
As far as quality of your data goes, unless your crystal is suffering from 
radiation damage (they all do!) from those free electrons/radicals
then your data should not be affected by the bleaching.
HTH.
Yuri



RHYS GRINTER wrote:

Hi All,

I was collecting some data at home on a crystal from a protein containing a 
ferredoxin domain. This crystal was originally red-brown in colour, but after 
16h of data collection, a large portion of it appears not be colourless. The 
crystal still seems to diffract fine (beyond 2A).
I assume this it due in some way to the radiation exposure but was wondering 
out curiosity whether someone had experienced this before and had a specific 
explanation for it. And if this will effect the integrity of the data.

Cheers

Rhys Grinter
PhD Candidate
University of Glasgow

[ccp4bb] Another lame question

[bhaba krishna Das]

I have sent this same query to JASCO earlier and did not get a response. So
thought of posting it here.

*Can a JASCO 810 CD spec be used as a simple UV-Vis Spec?*

I did not have a faster Spectrophotometer and so went ahead using JASCO 810
for a absorbance based enzyme assay/kinetics @ a constant wavelength,
without using the CD(mdeg) data.

The problem now is, although i found the data useful, i ain't sure if i can
use it, coz i have not found a reference yet!

-- 
Bhaba Krishna Das
Graduate Student
Structural and Computational Biology
ICGEB
ND-67

Re: [ccp4bb] Nucleophilic attack by the side-chain carboxyl group of Asp?

[Peter Hsu]

I too am also studying the reaction mechanism of an enzyme, but my 
chemistry/enzymatic biochemistry is rather weak after many years of non-use and 
no review. Does anyone know just as a general rule which residues are the best 
to worst nucleophiles?

Sorry if this seems rather presumptuous, just not sure where to look in 
literature for a summary of these things.